Biological contraceptive and contraceptive technique for males

ABSTRACT

A male contraceptive preparation and a contraceptive technique are disclosed. The contraceptive preparation consists essentially of 1,2,3-trihydroxypropane (&#34;THP&#34;; glycerol) in solution in a suitable carrier such as distilled water or saline solution. In accordance with the contraceptive technique, the contraceptive preparation is injected into the testes. The THP has been found to act as a potent inhibitor of spermatogenesis, resulting in long term infertility, with no apparent effect on libido, secondary sex characters, mating behavior and hormone levels. There are no observed side effects and no observed effects on other aspects of reproductive and hormonal biology.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to contraception, and specifically to abiological contraceptive and contraceptive technique for males.

In the case of humans, overpopulation remains one of the most criticalproblems facing humankind. In recent times, the contraceptive pill hasbeen successfully employed by women to prevent ovulation and conception.However, the contraceptive pill designed for use by women consists ofsteroids (a mixture of progestagens and estrogens) and there isconsiderable concern about its potentially harmful side-effectsfollowing prolonged use. There is thus at present considerable pressureto block the reproductive capacity in the male and to not place all thechemical and psychological burden on the female.

Methods of male fertility control are also highly desireable in the caseof animals, especially as simpler alternatives to the present proceduresof castration or other forms of surgical sterilization.

An ideal chemical contraceptive for the male would be one whicheffectively arrests spermatogenesis (i.e. stops formation of spermcells) or blocks the fertilizing capacity of sperm, without affectingtesticular steroidogenesis (production of male steroid hormones),libido, accessory sex glands, and pituitary function, in the absence oftoxic or other undesireable side effects. The procedure would be simple,non-surgical, and preferably expose only the sperm producing tissue tothe compound, thus avoiding distribution of foreign chemicals tonon-reproductive tissues in the body. Infrequent application of thetreatment would also be desirable.

2. Description of the Prior Art

An "ideal" male contraceptive agent has not been described in the priorart, although many compounds have been explored for the purpose ofinhibiting or arresting spermatogenesis (see Bennett, J. P. (1974),Chemical Contraception, Columbia Press, New York, pp. 133-170; Davies,A. G. (1980), Effects of Hormones, Drugs and Chemicals on TesticularFunction, Vol. 1, Eden Press, Westmount, pp. 123-164; and Jeffcoate, S.L. and Sandler, M. (Editors), Progress Towards a Male Contraceptive,John Wiley and Sons, Chichester, 1982). While many of the compoundstested in the past possess some anti-spermatogenic or anti-fertilizingcapacity, their contraceptive action is invariably overshadowed by theircytotoxic, neurotoxic or anti-metabolic effects, or by their untowardeffects on libido, accessory sex glands and the male endocrine system(see Jeffcoate and Sandler, supra). Moreover, the compounds that havebeen tested generally require fairly large daily doses and their longrange effects remain unknown (see Shandilya, L., Clarkson, T. B, Adams,M. R., and Lewis, J. C. (1982), "Effects of gossypol on reproductive andendocrine functions of male cynomolgus monkeys (Macaca fascicularis)",Biol.Reprod. 27:241-252).

Vasectomy is of course a proven technique, but it requires surgery,albeit relatively minor surgery. Epididymal sperm congestion and spermcysts and sometimes autoimmune reactions are observed.

Although castration will effectively remove sperm production, it alsoremoves the source of male sex steroid hormones and as such isunacceptable for human males. Castration is often employed on pets andfarm animals, but it requires aseptic surgery and a period of healingand thus is relatively complicated.

SUMMARY OF THE INVENTION

It is an object of the invention to provide a contraceptive andcontraceptive technique for males which offers advantages over thoseknown in the prior art.

In accordance with the present invention there is provided acontraceptive preparation intended for injection into the testes,consisting essentially of 1,2,3-trihydroxypropane ("THP"; glycerol) in asolution.

There is further provided a method of contraception comprising preparingthe solution, and then injecting a quantity into the testes.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described in greater detail, with reference totables included in the specification and with reference to theaccompanying drawings, in which:

FIG. 1 is a set of photographs of cross-sections of seminiferous tubulesfrom testes of control (FIG. 1 (a)) and treated (FIG. 1 (b)) rats;

FIG. 2 is a bar graph illustrating the effect of the treatment on tissueweights;

FIG. 3 is a bar graph showing the number of sperm in the epididymidesbefore and after mating of treated and untreated (control) rats;

FIG. 4 is a bar graph showing the effect of the treatment on serumhormone levels;

FIG. 5 is a bar graph showing the effect on libido; and

FIG. 6 is a graph illustrating the effect of the treatment on fertility.

DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention involves a single intratesticular injection with asolution comprised of components found in most living cells, i.e.components which are not foreign to the body, and are non-toxic andnonhormonal substances.

The preferred procedure is essentially as follows. A solution isprepared consisting of saline/1,2,3-trihydroxypropane ("THP"; glycerol)(3:7), or saline/THP/ethanol (3:7:0.25), or distilled water/THP (3:7),or distilled water/THP/ethanol (3:7:0.25). The proportions of water toTHP can be varied, as it is the THP which has the antispermatogenicaction.

The solution is then sterile filtered (0.2 micron filter units) intosterile serum vials. The scrotal area is cleaned with 95% alcohol.Injections are then made into the approximate center of the testis.

In rats, 50 to 200 microliters per testis has been found to besufficient to block spermatogenesis for at least 21 weeks. A total often experiments comprising over 200 rats and one experiment with 24hamsters have been conducted to test the effectiveness of the procedure.Preliminary conclusions from the most recent experiments are that anequivalent of 25 microliters at the above ratios is also effective, andexperimentation involving still lower concentrations is in progress.

EXAMPLES

Sprague-Dawley rats were injected intratesticularly with asterile-filtered solution of either A (THP/double distilledwater/ethanol, 7:3:0.25) or B (double distilled water or water/ethanol),the former rats being hereinafter referred to as "Treateds" and thelatter as "Controls". For the treatments, the rats were lightlyanesthetized with ether, the scrotum was wiped with alcohol, and thesolution was injected directly into the approximate center of the testesvia a gauge 27 needle. Histological, biochemical and fertilitydeterminations were made periodically after the injections.

In one experiment, 50 microliters of solution A was injected into theright testis of 48 day old rats. A week later, the injection wasrepeated. After one more week, i.e. two weeks after the first injection,the weights of the right testes were significantly less than the weightsof the left testes (e.g. 0.415±0.028 g. versus 1.039±0.067 g.).Histologically, the seminiferous tubules in the right testes were devoidof germinal cells but the interstitial cells appeared normal. Thereduced weights persisted at least 11 weeks after treatment.

In several subsequent experiments, the testes of 88-97 day old rats wereinjected once a week for 5 weeks, both with either solution A or B.Steroidogenesis, steroid enzyme activity, secondary sex characters,testicular histology and sexual behaviour were examined one week afterthe third and fifth injections and four weeks after the fifth injection.Homogenates from A-treated testes metabolized significantly more14C-progesterone into 14C-labelled 17alpha-OH-progesterone,20-alpha-dihydroprogesterone, androstenedione and testosterone on a permilligram protein basis than the B-treated ones. However, thesteroidogenic activity on a per testis basis did not differ betweenTreateds and Controls. Seminal vesicles and prostate weights were notaffected by the treatments, although the ratios of gonad to body weights(×100) were reduced from 0.67±0.06 in Controls to 0.34±0.03 in Treateds.Treateds and Controls showed the same level and degree of matingbehaviour when caged with virgin females. Four weeks after the fifthinjection, the epididymal sperm counts numbered 9.9×10⁵ (±3.5×10⁵) and7.5×10⁷ (±5.3×10⁷) in the Treateds and Controls respectively.

In a number of subsequent experiments, rats were given only a singleinjection into each testis and histological, biochemical, and fertilitydeterminations were made for up to 21 weeks after the injection. Theresults showed that within one week of the injection with THP,spermatogenesis had been arrested and within 2 weeks the seminiferoustubules were largely depleted of spermatogenic (sperm producing) cells;they remained devoid of dividing germ cells for the remainder of theexperimental period. On the other hand, the Leydig (androgen producing)cells had a normal morphological appearance. Histochemical andbiochemical studies showed that the activities of steroid enzymes(3-beta-hydroxysteroid dehydrogenase, 3-alpha-hydroxysteroiddehydrogenase, 17-beta-hydroxysteroid oxidoreductase,17-alpha-hydroxylase, and C₁₇₋₂₀ -lyase) involved in the androgenproduction, the in vitro conversion of ¹⁴ C-progesterone, and theproduction of testosterone and androstenedione were not altered by theTHP treatment. Similarly, the blood serum levels of testosterone andpituitary gonadotropins (LH and FSH), and the weights of theandrogen-dependent prostate gland and seminal vesicles were the same asin the Controls. THP treated males showed the same level of sexualbehaviour and mated with virgin females at the same frequency asControls but all were infertile after the 3rd mating and remained 100%infertile for the duration of the experiments (21 weeks). The totalnumber of sperm stored in the epididymides of treated rats declinedrapidly and was reduced by 99.99% (of Controls' numbers) after the thirdmating.

The results of a number of such experiments will now be set out ingreater detail.

Table 1 below shows the effects of THP treatment on the weights oftestes, epididymides, prostate and seminal vesicles, and on the numberof sperm in the epididymides, from one of the experiments. The resultswere similar in each of the other experiments where time of treatment,age of rats, and number of injections had been different. Table 1 showsthat the weights of the Treated testes were significantly reduced oneweek after a single injection, and had declined to 46% and 37% of theControl testes weights by 2 and 11 weeks respectively. In the sameexperiment, the weights of the epididymides were significantly reducedin animals sampled 1, 2, and 11 weeks after the injection. The averagenumber of sperm in each epididymis, following three matings, was reducedby 99.99% in the Treated animals. On the other hand, the weights of theaccessory sex structures (prostate and seminal vesicles) were notaffected by the treatment.

                  TABLE 1                                                         ______________________________________                                        Effects of THP Treatment on Weights of Testes and                             Accessory Structures and on Epididymal Sperm Number                                     Weeks after                                                         Tissue    injection  Control(g)  Treated(g)                                   ______________________________________                                        Testis     1         3.24 ± .25                                                                             2.07 ± .19                                           2         3.35 ± .06                                                                             1.55 ± .07                                          11         3.31 ± .12                                                                             1.24 ± .04                                Epididymis                                                                               1         1.33 ± .13                                                                              .92 ± .04                                           2         1.33 ± .09                                                                              .93 ± .07                                          11         1.35 ± .07                                                                              .91 ± .08                                Prostate  11         0.83 ± .08                                                                              .81 ± .10                                Seminal vesicle                                                                         11         1.55 ± .07                                                                             1.52 ± .09                                Total sperm                                                                             11         25.85 × 10.sup.7                                                                    9.9 × 10.sup.3                         in epididymis        (±2.69 × 10.sup.7)                                                               (±6.8 × 10.sup.3)                   after 3 matings                                                               ______________________________________                                         Twenty-four male rats, 85-91 days old (325-350 g) at the start of the         experiment. Each testis injected with 200 microliters of sterilefiltered      distilled water (Controls) or THP/water (7:3) (Treated) on day one.           Animals were sampled 1, 2, and 11 weeks later. Each value represents the      mean ± SEM obtained from tissues of 3-5 animals.                      

The photographs of FIG. 1 show the suppressive effects of THP treatmenton gametogenesis. The effect appears to be rapid and highly selective,so that just 2 weeks after an injection, many seminiferous tubules areessentially empty of spermatogenic cell stages and spermatozoa, beinglined only with normal appearing Sertoli cells and occasional primarygerm cells. The interstitial Leydig cells, on the other hand, which arethe primary source of testicular androgens, do not appear to be affectedby the treatment. Both photographs are cross-sections of seminiferoustubules from rat testes, with a magnification of 180, 14 days afterinjection with distilled water (FIG. 1a) and THP/water (7:3) (FIG. 1b).Different stages of spermatogenic cells S are seen within the tubulesand spermatozoa reside in the lumen (arrow), indicating normalprogresssion of spermatogenesis in the control testes (FIG. 1a). Thetubules from the treated testes (FIG. 1b) are devoid of spermatogeniccells, being lined only with Sertoli cells and some primary germ cells.The apparent increase in interstitial cells I is probably due to themarked decrease in seminiferous tubule diameter. Steroidogenic studiessupport the view that there has not been an increase in Leydig cellnumber nor a change in total steroidogenic activity.

FIG. 2 is a bar graph representation of typical experimental results,showing the effect of the treatment on tissue weights in milligrams pergram of body weight.

FIG. 3 is a bar graph representation of typical epididymal sperm countsof treated and control animals before and after matings.

The two primary functions of the testes are spermatogenesis andsteroidogenesis. Although spermatogenesis was markedly suppressed by theTHP treatment, Table 2 below shows that steroidogenesis was notsignificantly affected. The activities of the four major enzymes shownin the table increased significantly when calculated on a per mg proteinbasis. However, when calculated on a per gonad basis, there are nosignificant differences in activities between Control and Treatedtestes.

                                      TABLE 2                                     __________________________________________________________________________    Effect of THP Treatment on Steroidogenesis                                    Steroid Microunits/mg protein                                                                        Microunits/testis pair                                 enzyme  Control                                                                              Treated Control                                                                              Treated                                         __________________________________________________________________________    20-alpha-HSD                                                                           8.64 ± 1.33                                                                      19.67 ± 2.01                                                                       1465 ± 224                                                                        1475 ± 150                                   17-alpha-                                                                             27.22 ± 4.68                                                                       61.91 ± 18.82                                                                     4600 ± 790                                                                         4643 ± 1411                                 hydroxylase                                                                   C.sub.17-20 -                                                                         16.60 ± 3.31                                                                      38.19 ± 9.11                                                                       2805 ± 559                                                                        2864 ± 683                                   lyase                                                                         17-beta-HSD                                                                           15.29 ± 2.96                                                                      28.37 ± 5.69                                                                       2584 ± 500                                                                        2127 ± 427                                   Androgen                                                                               0.61 ± 0.32                                                                      1.33 ± 0.3                                                                         101.4 ± 28.9                                                                      107.9 ± 24.3                                 produced                                                                      __________________________________________________________________________     Sixteen male rats (96-100 days old) were used for this study. Half were       injected intratesticularly with sterilefiltered distilled water (Control)     and half with THP/water (7:3) solution (Treated). Nine weeks later the        gonads were removed and homogenized; and enzyme activity was determined.      One microunit equals 1 picomole of steroid converted or produced per          minute. Androgen produced is the total .sup.14 Clabelled testosterone and     androstenedione produced by homogenate in 30 minutes.                    

As can be seen from Table 3 below, the serum levels of LH and FSH werenot significantly altered by the THP treatment, in contrast tocastration which resulted in marked increases. This is also illustratedin FIG. 4.

                  TABLE 3                                                         ______________________________________                                        Effect of THP treatment on                                                    serum LH, FSH, and testosterone levels                                                                 Castrated                                                                              Young                                       Control       Treated    Males    Females                                     ______________________________________                                        Testost-                                                                             5.01 ± 1.4                                                                             5.21 ± 1.08                                                                          N.D.   N.D.                                      erone                                                                         LH      97.5 ± 25.9                                                                          86.7 ± 8.7                                                                            560     33.5 ± 16.8                           FSH    197.5 ± 21.9                                                                          255.0 ± 38.1                                                                          740    192.2 ± 35.9                           ______________________________________                                         Serum was collected from Control and Treated rats described in Table 2.       Values for Control and Treated rats are means ± SEM (n = 3). Values fo     castrated males are the means for two adult rats. Values for young female     are means ± SEM (n = 9) for 21day old female rats. All values are in       ng/ml serum. ND = not determined.                                        

The amount of androgen (testosterone and androstenedione) produced invitro from progesterone by the testes (Table 2) and the amounts of serumandrogen (Table 3) are the same in Treated and Control animals, and theweights of the androgen-dependent prostates and seminal vesicles werenot changed (Table 1).

Mating studies showed that Treated males exhibit the same level ofsexual behaviour as the Controls, when caged with virgin females, andthat they mate with the same frequency, as shown in Table 4 below and inFIG. 5. However, the fertility of the Treated males declined markedly bythe 3rd mating and all Treated males were shown to be infertile by the4th mating, as shown in Table 4 and FIG. 6. The fertility of the Treatedmales following the initial matings may be explained by the presence ofviable spermatozoa stored in the epididymides. Since the spermatozoa cannot be replenished by the spermatogenetically inactive testes, theanimals are infertile after the stored sperm in the epididymides aredepleted, following several matings.

Periods 1 through 5 in Table 4 and in FIG. 6 represent 2 through 6 weeksrespectively after injection. However, there was a substantial lapse oftime thereafter, prior to periods 6 and 7 in FIG. 6. Periods 6 and 7represent 20 and 21 weeks respectively after injection, indicating noresumption of fertility even after 21 weeks.

                  TABLE 4                                                         ______________________________________                                        Effect of THP Treatment on Mating Frequency and on Fertility                         Control       Treated                                                  Period of                                                                              Number    Number    Number  Number                                   cohabitation                                                                           mated     pregnant  mated   pregnant                                 ______________________________________                                        1        7         6         7       6                                        3        8         7         8       2                                        4        7         7         9       0                                        5        8         8         8       0                                        ______________________________________                                         Male rats (n = 10), 80-90 days old at the start of the experiment, were       treated as in Table 1, with either water (Control) or THP/water (7:3)         solution (Treated). Two weeks later a virgin female rat was placed with       each male for a period of 5 days (Monday to Friday). The appearance of a      vaginal plug was taken as evidence that mating had occurred. The procedur     was repeated with fresh virgin females on 5 successive weeks (Periods         1-5). Pregnancy was assessed by examination of uteri 10 days after the en     of each cohabitation.                                                    

It was concluded as a result of the experiments that THP, when appliedintratesticularly, acts as a potent inhibitor of spermatogenesis,resulting in long term infertility without affecting libido, secondarysex characters, mating behaviour and hormone levels. This treatmentexhibits specific and potent antispermatogenic action with no observedside-effects and no observed effects on other aspects of reproductiveand hormonal biology.

The treatment is very selective for spermatogenesis, with no reductionin male hormone production, in secondary sex characteristics, libido,sexual arousal and behaviour and no significant alteration in serumhormone levels. The treatment appears to be highly specific for thegerminal cells; according to all histological, histochemical andbiochemical data, the steroid hormone producing cells in the testisremain unaffected by the treatment.

In contrast to vasectomy, the procedure requires no surgery andtherefore would seem to be more suitable as a technique for massapplication. Also, since it halts spermatogenesis, this procedure willnot promote the epididymal sperm congestion, sperm cysts, and autoimmunereactions observed with vasectomy.

In contrast to castration, aseptic surgery is avoided, and there is noperiod of healing. Also, there is no removal of the source of male sexsteroid hormones as is the case with castration.

It is not yet known if the treatment is reversible, and it has not yetbeen verified that the treatment is effective in all mammals.Experiments in progress with hamsters indicate that the treatment isjust as effective in this species as in the rat: within two weeks aftera single injection, weights of the testes and epididymides were reducedby 37 to 43 percent, and the number of sperm stored in epididymides wasdecreased by 75 percent in the THP treated, unmated hamsters.Experiments on rabbits and primates are to follow, and are reasonablyexpected to show equally successful results.

It will be appreciated that the above description of the invention is adescription of the state of knowledge to date with respect to theinvention. Examples are given for the purpose of illustration only. Manyvariations on the details of the description may be possible,particularly but not exclusively with respect to solution strengths anddosage levels. Such variations are clearly envisioned as being withinthe spirit and substance of the scope of the invention as defined andclaimed, whether or not expressly claimed, since these variations may bedeveloped through routine non-inventive experimentation given theteachings of this disclosure.

What is claimed as the invention is:
 1. A method of male contraception comprising injecting a suitable quantity of 1,2,2-trihydroxypropane in a suitable pharmaceutical carrier into the testes.
 2. A method of male contraception as recited in claim 1, in which said carrier is distilled water.
 3. A method of male contraception as recited in claim 2, in which the ratio of distilled water to 1,2,3-trihydroxypropane is approximately 3:7.
 4. A method of male contraception as recited in claim 1, in which said carrier is a 0.9 percent saline solution.
 5. A method of male contraception as recited in claim 4, in which the ratio of saline solution to 1,2,3-trihydroxypropane is approximately 3:7.
 6. A method of male contraception as recited in claim 1, in which said carrier is a 0.9 percent saline solution and ethanol in a saline to ethanol ratio of approximately 12:1.
 7. A method of male contraception as recited in claim 6, in which the ratio of saline to 1,2,3-trihydroxypropane to ethanol is approximately 3:7:0.25.
 8. A method of male contraception as recited in claim 1, in which said carrier is distilled water and ethanol in a distilled water to ethanol ratio of approximately 12:1.
 9. A method of male contraception as recited in claim 8, in which the ratio of distilled water to 1,2,3-trihydroxypropane to ethanol is approximately 3:7:0.25. 